Membrane-type MMPs are indispensable for placental labyrinth formation and development. We have demonstrated that MT1-MMP deficiency is compatible with development, however, postnatal survival and growth is substantially reduced due to the loss of ability to turn over collagen matrices in connective tissues. These observations, while illustrating the importance of collagen remodeling, also highlights that MT-MMP function is redundant and we have systematically dissected the function of MT1-MMP, MT2-MMP and MT3-MMP. From these studies, we have previously found that MT3-MMP works in concert with MT1-MMP. Combined loss of MT1-MMP and MT3-MMP leads to demise immediately following birth. We therefore reasoned that another MT-MMP family member with a wider expression pattern could be responsible for the survival of the mice up to birth, or that MT-MMP activity simply is dispensable prior to birth. To test this hypothesis, we generated mice that are MT2-MMP deficient under the assumption that MT2-MMP could compensate for the loss of MT1-MMP or MT3-MMP during embryonic development. MT2-MMP deficient mice are healthy and grossly indistinguishable from wild type littermates. To determine if the combined activity of MT-MMPs is required for development, we crossed the MT2-MMP deficient mouse strain to the MT1-MMP deficient mouse strain. Intercrossing of mice doubly heterozygous for the two MT-MMPs failed to produce offspring with combined MT1-MMP/MT2-MMP deficiency. To account for this apparent deficit in greater detail, we collected embryos from timed pregnant female mice and established that double deficient offspring were present at E9, but subsequently involuted between E10.5 and E11.5. Closer inspection of the embryos revealed signs of placental defects manifested by grossly dilated vessels. Notably, electron microscopy analysis of the placenta revealed a failure to establish the two-syncytiotrophoblast layers around the embryonic vessels, which are required for normal labyrinth formation. Consequently, we explored the functions of MT1-MMP and MT2-MMP in the process of placental morphogenesis. The fetal portion of the placenta, in particular the labyrinth, displays strong overlapping expression of MT1-MMP and MT2-MMP, which is critical for syncytiotrophoblast formation and in turn for formation of fetal vessels. Disruption of trophoblast syncytium formation consequently lead to developmental arrest with only a few poorly branched fetal vessels entering the labyrinth, causing death at embryonic day 11.5. Based on the lack of syncytiotrophoblast formation, our data suggested that MT-MMP activity in the placenta affected the cell fusion processes required for this syncytia formation. Through knockdown of conditional MT2-MMP expression in the MT1-MMP null mice, we demonstrated that MT-MMP is crucial specifically during development of the labyrinth. In contrast, knockdown of conditional MT2-MMP activity after labyrinth formation is compatible with development to term and postnatal life. Taken together these data identify essential but interchangeable roles for MT1-MMP or MT2-MMP in placental vasculogenesis and provide the first example of selective temporal and spatial MMP activity required for development of the mouse embryo. Remodeling of unmineralized cartilages by MT-MMPs We have previously accounted for the role of MT-MMP activity in remodeling of specific unmineralized cartilages in the skeleton. Until now, it has remained unresolved if this remodeling mechanism was a property of chondrocytes or connective tissue cells adjacent to the cartilage. Using a cartilage-specific ablation of MT-MMP activity, we have established that chondrocytes are essential for the remodeling of these specific cartilages. Importantly, neighboring cells fail to remodel these unmineralized cartilages and chondrocytes are thus essential for this remodeling process. Because unmineralized cartilage is frequently destroyed in pathological conditions, we suggest an important role for MT-MMP activity in pathological joint disease that identify a potential effective drug target to prevent cartilage destruction. The role of MT-MMP expression in specific cell types associated with the skeleton Having assigned the global role of MT-MMP activity, we have initiated a refined dissection of the cell and tissue specific role of the MT-MMP function in skeletal development and homeostasis. We are specifically focusing on the role of protease-mediated cellular signaling and matrix processing in the skeleton and hematopoietic cell environment using a cell- and tissue-specific ablation strategy to assign function of MT-MMPs. Our initial observations have pointed to a pivotal role of MT-MMP activity in the control of bone resorption. Specifically, loss of MT-MMP activity leads to rampant loss of bone and we have pursued the possibility that protease-mediated attenuation of osteoclast differentiation directed by RANKL-signaling is mediated by shedding of this molecule. We have recently demonstrated that osteoblast and pre-osteoblastic loss of MT1-MMP expression leads to the same increase in osteoclast differentiation as observed in mice with global loss of MT1-MMP. Furthermore, we demonstrated that MT1-MMP specifically cleaves RANKL and thus interplays with the RANKL decoy, OPG, in attenuation of RANKL signaling through the RANK receptor on osteoclast precursors. Additionally we have investigated the role of MT-MMP activity in vascular smooth muscle cell and pericyte-like cells by selective ablation of MT-MMP activity in SM22 positive cells. While mice with this specific loss of MT-MMP activity display relatively normal bone resorption, a marked reduction in bone formation is evident. Additionally, prominent fibrosis is present in the cranial vault and sutures display inability to properly form. These results point to a pivotal role for MT-MMP activity in perivascular progenitor cells, which during development and postnatal growth, are recruited into connective tissues and differentiate there into skeletal cells. MT-MMP contributions to the hematopoietic microenvironment and homeostasis Because the hematopoietic environment is critical for proper bone formation due to the contribution of hematopoietic cells to bone remodeling, we have focused on the role of MT-MMP activity in this tissue compartment as well. Notably, skeletal cells devoid of MT-MMP activity fail to support postnatal marrow formation in ectopic bone formation. We propose that MT-MMPs play a pivotal role in the migration and recruitment of hematopoietic stem cells and their ability to populate a potential hematopoietic environment. We have demonstrated thus far that the bone marrow of MT1-MMP deficient mice retain twice as many hematopoietic stem cells (HSCs) as observed in wildtype littermates. In an effort to identify the HSC-retaining cell and the cell responsible for release of HSCs to the vasculature, we are currently analyzing cell-specific ablation strains for their marrow-associated HSC count, and their to ability to release these cells into the circulation following chemically-induced mobilization of HSCs.